Heavy chain diversity region segments of the channel catfish: Structure, organization, expression and phylogenetic implications

Circular DNA, derived from lymphocytes of juvenile channel catfish, was use d to construct A libraries that were screened to identify the products of i mmunoglobulin D-H-J(H) excision events. Clones were characterized that cont ained D-H to J(H) recombination signal joints. The signal joints represente d 23-bp recombination signal sequences (RSS) identical to germline J(H) seg ments that were adjacent to D-H 12-bp RSS elements. D-H flanking regions wi thin the clones were used to probe a genomic library. Three germline D-H ge ne segments containing 11-19 bp coding regions flanked by 12-bp RSS element s with conserved heptamers and nonamers were identified. The DH locus is cl osely linked to the J(H) locus, and Southern blots indicate that the D-H se gments represent different single member gene families. Analysis of H chain cDNA shows that each germline D-H segment was expressed in functional VDJ recombination events involving different J(H) segments and members of diffe rent V-H families. Several aspects of CDR3 junctional diversity were eviden t, including deletion of coding region nucleotides, N- and P-region nucleot ide additions, alternate D-H reading frame utilization, and point mutations . Coding region motifs of catfish D-H segments are phylogenetically conserv ed in some D-H segments of higher vertebrates. These studies indicate that the structure, genomic organization, and recombination patterns of D-H segm ents typically associated with higher vertebrates evolved early in vertebra te phylogeny at the level of the bony fish.